RNAs that do not function as messenger RNAs, transfer RNAs or ribosomal RNAs, are collectively termed non-coding RNAs (ncRNAs). ncRNAs can range in size from 21-25 nucleotides (nt) up to >10,000 nt, and estimates for the number of ncRNAs per genome range from hundreds to thousands. The functions of ncRNAs, although just beginning to be revealed, appear to vary widely from the purely structural to the purely regulatory, and include effects on transcription, translation, mRNA stability and chromatin structure (G. Storz, Science (2002) 296: 1260-1262). Two recent pivotal discoveries have placed ncRNAs in the spotlight: the identification of large numbers of very small ncRNAs of 20-24 nucleotides in length, termed micro RNAs (miRNAs), and the relationship of these miRNAs to intermediates in a eukaryotic RNA silencing mechanism known as RNA interference (RNAi).
RNA silencing refers to a group of sequence-specific, RNA-targeted gene-silencing mechanisms common to animals, plants, and some fungi, wherein RNA is used to target and destroy homologous mRNA, viral RNA, or other RNAs. RNA silencing was first observed in plants, where it was termed posttranscriptional gene silencing (PTGS). Researchers, trying to create more vividly purple flowers, introduced an extra copy of the gene conferring purple pigment. Surprisingly, the researchers discovered that the purple-conferring genes were switched off, or cosuppressed, producing white flowers. A similar phenomenon observed in Fungi was termed quelling. These phenomena were subsequently found to be related to a process in animals called RNA interference (RNAi). In RNAi, experimentally introduced double-stranded RNA (dsRNA) leads to loss of expression of the corresponding cellular gene. A key step in the molecular mechanism of RNAi is the processing of dsRNA by the ribonuclease Dicer into short dsRNAs, called small interfering RNAs (siRNAs), of ˜21-23 nt in length and having specific features including 2 nt 3′-overhangs, a 5′-phosphate group and 3′-hydroxyl group. siRNAs are incorporated into a large nucleoprotein complex called RNA-induced silencing complex (RISC). A distinct ribonuclease component of RISC uses the sequence encoded by the antisense strand of the siRNA as a guide to find and then cleave mRNAs of complementary sequence. The cleaved mRNA is ultimately degraded by cellular exonucleases. Thus, in PTGS, quelling, and RNAi, the silenced gene is transcribed normally into mRNA, but the mRNA is destroyed as quickly as it is made. In plants, it appears that PTGS evolved as a defense strategy against viral pathogens and transposons. While the introduction of long dsRNAs into plants and invertebrates initiates specific gene silencing (3,4), in mammalian cells, long dsRNA induces the potent translational inhibitory effects of the interferon response (8). Short dsRNAs of <30 bp, however, evade the interferon response and are successfully incorporated into RISC to induce RNAi (9).
Another group of small ncRNAs, called micro RNAs (miRNAs), are related to the intermediates in RNAi and appear to be conserved from flies to humans (2, 12, 13). miRNAs are putatively transcribed first as a long transcript (pri-miRNAs), in some cases as miRNAs clusters, and these transcripts are then processed to ˜70 nt RNA precursors (pre-miRNAs) having a predicted stem-loop structure. The enzyme Dicer cleaves the pre-miRNAs to produce ˜20-24 nt miRNAs that function as single-stranded RNAi mediators (4, 10). These small transcripts have been proposed to play a role in development, apparently by suppressing target genes to which they have some degree of complementarity. The founding members of miRNAs, lin-4 and let-7, exert their control of gene expression by binding to non-identical sequences in the 3′ UTR of mRNA, thereby preventing mRNA translation (17). In recent studies, however, miRNAs bearing perfect complementarity to a target RNA could function as siRNAs to specifically degrade the target sequences (14, 15). Thus, the degree of complementarity between an miRNA and its target may determine whether the miRNA acts as a translational repressor or as a guide to induce mRNA cleavage.
The discovery of miRNAs as endogenous small regulatory ncRNAs may represent the tip of the iceberg, with other groups of regulatory ncRNAs still to be discovered. Meanwhile, RNAi is now poised to revolutionize reverse genetics approaches, enabling virtually any gene of interest to be disrupted quickly and efficiently. Limitations of current RNAi technologies include their dependence upon inefficient transfection techniques and intrinsically transient nature. A challenge that must be met to realize the promise of future RNAi-based therapeutics is the development of efficient systems for siRNA delivery and expression in mammalian cells and organisms.